The present invention relates generally to materials and methods useful in the quantitative detection and affinity purification of human immune interferon ("IFN-.gamma.") and structurally related polypeptides such as analogs of INF-.gamma. produced by recombinant methods.
Of substantial interest to the background of the present invention is the state of the art with regard to the preparation and use of a class of biologically active substances, the interferons (IFNs). Interferons are secreted proteins having fairly well-defined antiviral, antitumor and immunomodulatory characteristics. See, e.g., Gray, et al., Nature, 295, pp. 503-508 (1982) and Edge, et al., Nature, 292, pp. 756-782 (1981), and references cited therein.
On the basis of antigenicity and biological and chemical properties, human interferons have been grouped into three major classes: IFN-.alpha. (leukocyte), IFN-.beta. (fibroblast) and IFN-.gamma. (immune). Considerable information has accumulated on the structures and properties of the virus-induced acid-stable interferons (IFN-.alpha. and .beta. ). These have been purified to homogeneity and at least partial amino acid sequences have been determined. Analyses of cloned cDNA gene sequences for IFN-.beta. and the IFN-.alpha. multigene family have permitted the deduction of the complete amino acid sequences of many of the interferons. In addition, efficient synthesis of IFN-.beta. and several IFN-.alpha.s in E. coli, and subtype IFN-.alpha..sub.1, in yeast, have now made possible the purification of large quantities of these proteins in biologically active form.
Much less information is available concerning the structure and properties of IFN-.gamma., an interferon generally produced in cultures of lymphocytes exposed to various mitogenic stimuli. It is acid labile and does not cross-react with antisera prepared against IFN-.alpha. or IFN-.beta.. A broad range of biological activities have been attributed to IFN-.gamma. including potentiation of the antiviral activities of IFN-.alpha. and -.beta. from which it differs in terms of its virus and cell specificities and the antiviral mechanisms induced. In vitro studies performed with crude preparations suggest that the primary function of IFN-.gamma. may be as an immunoregulatory agent. The antiproliferative effect of IFN-.gamma. on transformed cells has been reported to be 10 to 100-fold greater than that of IFN-.alpha. or -.beta., suggesting a potential use in the treatment of neoplasia. Murine IFN-.gamma. preparations have been shown to have significant antitumor activity against mouse sarcomas.
It has recently been reported (Gray, et al., supra) that a recombinant plasmid containing a cDNA sequence coding for human IFN-.gamma. has been isolated and characterized. Expression of this sequence in E. coli and cultured monkey cells is reported to give rise to a polypeptide having the properties of authentic human IFN-.gamma.. In the publication, the deduced 146 amino acid sequence of the "mature" polypeptide, exclusive of the putative leader sequence, is as follows: ##STR1##
In a previous publication of the sequence, arginine, rather than glutamine, was specified at position 140 in the sequence. (Unless otherwise indicated, therefore, reference to "human immune interferon" or, simply "IFN-.gamma." shall comprehend both the [Arg.sup.140 ] and [Gln.sup.140 ] forms.)
The above-noted wide variations in biological activities of various interferon types makes the construction of synthetic polypeptide analogs of the interferons of paramount significance to the full development of the therapeutic potential of this class of compounds. Co-owned, co-pending U.S. patent application Ser. No. 375,494 by Alton, et al., filed May 6, 1982, relates to procedures for the rapid manufacture of structural genes specifying human interferons and analogs thereof and to the use of these manufactured genes to secure microbial expression of biologically active polypeptide products.
Of substantial interest to the background of the invention is the extensive body of information generated over the recent past with respect to the use of immunological procedures for the isolation and quantitative detection of complex, biologically active polypeptides such as the inteferons. Such immunological procedures have frequently employed polyclonal, serum-derived antibodies directed against the proteinaceous materials of interest. Briefly put, antisera and serum-derived antibodies are obtained by inoculation of an immunologically-active animal, such as a rat or rabbit, with the material of interest. The injected material is recognized as a foreign antigenic substance by the immune system of the animal and elicits production of antibodies against the antigen. Differing cells responding to stimulation by the antigenic substance produce and release into circulation antibodies slightly different from those produced by other responding cells. The antibody activity remains in the serum of the animal when its blood is extracted. While unpurified serum or antibody preparations purified as serum immunoglobulin fractions may then be used in assays to detect and complex with the proteinaceous material in fluids and on gels, the antibody preparations suffer from a major disadvantage. Serum antibodies, composed of all the different antibodies produced by individual cells, are polyclonal in nature and for the most part are reactive with antigens other than the one of interest. Antibodies specific for the desired product represent a subset of the antibodies present in serum.
Of interest to the background of the present invention are recent advances in the art of developing continuous cultures of cells capable of producing a single species of antibody (a "monoclonal" antibody) which is specifically immunologically reactive with a single antigenic determinant of a selected antigen. See, generally, Chisholm, High Technology, Vol. 3, No. 1, 57-63 (1983) and Todd, et al., T.I.B.S., 7, pp. 212-216 (1982). Monoclonal antibodies have proven to be invaluable for the characterization, quantitative analysis, and purification of macromolecular antigens, including such biologically active compounds as leukocyte interferons. See generally, Secher, et al., Nature, 285, pp. 446-450 (1980); Staehelin, et al., P.N.A.S, 78, pp. 1848-1852 (1981); and Imai, et al., J.Immunology, 128, pp. 2824-2825 (1982). The lack of availability of monoclonal antibodies to IFN-.gamma. is becoming a more pronounced problem in view of the above-noted advances in the capacity to employ recombinant methods for the production of IFN-.gamma. and its analogs. Antibodies with high specific affinity for these substances would not only be useful for purification of IFN-.gamma. and analogs thereof from microbial production systems, but would also be useful in quantitative analytical methods involving IFN-.gamma.. For example, such antibody preparations would be useful in the characterization of cell surface receptors for IFN-.gamma. (including their distribution and modulation) and in characterizing the receptor binding qualities of IFN-.gamma. analogs. Such information, in turn, would have clinical diagnostic and therapeutic significance in interferon management of disease.
Also of interest to the background of the invention are reports of the immunological activity of synthetic peptides which substantially duplicate the amino acid sequence extant in naturally-occurring proteins, glycoproteins and nucleoproteins. More specifically, relatively low molecular weight polypeptides have been shown to participate in immune reactions which are similar in duration and extent to the immune reactions of physiologically significant proteins such as viral antigens, polypeptide hormones and the like. Included among the immune reactions of such polypeptides is the provocation of the formation of specific antibodies in immunologically active animals. See, e.g., Lerner, et al., Cell, 23, 309-310 (1981); Ross, et al., Nature, 294, 654-656 (1981); Walter, et al., P.N.A.S. (U.S.A.), 77, 5197-5200 (1980); Lerner, et al., P.N.A.S. (U.S.A.), 78, 3403-3407 (1981); Walter, et al., P.N.A.S. (U.S.A.), 78, 4882-4886 (1981); Wong, et al., P.N.A.S. (U.S.A.), 78, 7412-7416 (1981); Green, et al., Cell, 28, 477-487 (1982); Nigg, et al., P.N.A.S. (U.S.A.), 79, 5322-5326 (1982); Baron, et al., Cell, 28, 395-404 (1982); Dreesman, et al., Nature, 295, 158-160 (1982); and Lerner, Scientific American, 248, No. 2, 66-74 (1983).
The advantages of antibody preparations immunoreactive with both a macromolecule and with a readily available synthetic peptide are manifest. Co-owned, co-pending U.S. patent application Ser. No. 463,724, by Egrie, filed Feb. 4, 1983 discloses the development and use of monoclonal antibodies which are both immunologically reactive with human erythropoietin and reactive with a synthetic polypeptide which is duplicative of an amino acid sequence determined to be extant in erythropoietin. This achievement is indeed a rare one, owing to the fact that polypeptide fragments in solutions employed as inoculants cannot ordinarily be expected to assume an "antigenic" configuration similar to that which the polypeptide sequence naturally assumes as part of a macromolecular material. Consequently, monoclonal antibodies to whole macromolecules seldom bind peptide fragments well, and vice versa.
To date, there have been no published reports of the development of monoclonal antibodies, raised against interferon peptide fragments, and which have high affinity for interferon-duplicating peptide fragments and high affinity for native interferon species. It has quite recently been reported that polyclonal antibody preparations have been raised in rabbits in response to administration of a synthetic peptide corresponding to the first 20 amino acids of the amino (N) terminal of IFN-.gamma.. [Johnson, et aI., J. Immunology, 129, pp. 2357-2359 (1982).] Serum antibodies obtained according to the published procedure were noted to be operative in neutralizing the antiviral biological effects of IFN-.gamma.. While the capacity of antibodies to neutralize biological effects of macromolecules is in some instances considered to be an advantageous characteristic, this is not uniformly the case. As one example, neutralizing antibodies are of limited use in studies directed to elucidation of target cell receptor structure and function. More significantly, use of neutralizing antibodies in affinity purification procedures (wherein products are often isolated by quite harsh elution processes from bound association with fixed antibody preparations) may result in loss of biological activity of the purified material through disruption of "active" sites overlapping sites where binding to the antibody takes place.
There continues to exist a need in the art, therefore, for monoclonal antibodies which are specifically immunoreactive with IFN-.gamma.. In order to meet the needs of the art, monoclonal antibody preparations would advantageously be immunoreactive with native and recombinant IFN-.gamma. and analogs thereof, would also be immunoreactive with synthetic peptides substantially duplicative of IFN-.gamma., and would preferably be non-neutralizing of IFN-.gamma. biological activity.